Bacteriophage T4 codes for all the essential proteins involved in its DNA replication. At present, eleven gene products are implicated in the replication process. The functions of eight of these have been clearly identified (for a review see Nossal, N. G. and Alberts, B. M. (1983) in "Bacteriophage T4", eds. Mathews, C. K., Kutler, E. M., Mosig, G. and Berget, P. B. (Am. Soc. Microbiol., Washington, D.C.), pp. 71-81). They are products of gene 43 (DNA polymerase), gene 32 (helix destabilizing protein), genes 44 and 62 (a complex with DNA-dependent ATPase activity), gene 45 (stimulates 44/62 protein complex ATPase), gene 41 (helicase), gene 61 (RNA priming protein), and gene dda (delicase). DNA polymerase is absolutely required for the initiation and maintenance of viral replication. DNA polymerase was first identified as the product of gene 43 when mutants of the latter were shown to lack a functional polymerase (Warner, H. R. and Barnes, J. E. P (1966). Virology 28: 100-101).
The purified T4 polymerase is a monomer of about 110 kilodaltons. It catalyzes three reactions: (i) 5' to 3' polymerization on a primed single-stranded DNA template; (ii) 3' to 5' exonucleolytic hydrolysis of single-stranded DNA and of the 3'--OH end on duplex DNA at a slower rate; and (iii) primer-template dependent turnover of dNTP to dNMP (Goulian, M. Lucas, Z. J., and Kornberg, A. (1968), J. Biol Chem, 243:627-638; and Hershfield, M. S. and Nossal, N. G. (1972), J. Biol. Chem., 247: 3393-3404). The turnover activity appears to be a result of hydrolysis of newly incorporated nucleotides of the 3' terminus.
Gene 43 is transcribed from its own promoter into a monocistronic mRNA (Young, E. T. and Menard, R. C. (1981), J. Virol. 40: 772-789). The level of gene 43 production is self-regulated. T4 phage carrying mutations of gene 43 greatly overproduce a defective polymerase (Russel, M. (1973), J. Mol. Biol., 79: 83-94). This regulation occurs at the level of transcription (Krisch, N. H., van Houwe, G., Belin, D., Gibbs, W. Epstein, R. H. (1977), Virology, 78: 87-98). The amount of polymerase produced is also increased by mutations in genes 44 and 45, leading to the suggestion that autoregulation of gene 43 expression is enhanced when the polymerase is associated in a complex with at least gene products of 44 and 45 (Miller, R. C., Young, E. T., Epstein, R. H., Krisch, H. M., Mattson, T. and Bolle, T. A. (1981), Virology, 110:98-112).
T4 DNA polymerase is an ideal system for studies of the structure and function of a DNA polymerase. It also is an important enzyme for molecular biologists in the process of DNA manipulation. (See, e.g., Maniatis, infra.) Unfortunately, purification of T4 DNA polymerase from T4 infected E. coli cells is time consuming and provides only small amounts of pure protein (Morris, C. F., Hama-Inaba, H., Mace, D. Sinha, N. K., and Alberts, B. M. (1979), J. Biol. Chem., 254:6787-6796). Attempts to clone the entire gene 43 have not been successful. However, a large DNA fragment containing the NH-terminal of gene 43 has been cloned (Wilson, G. G., Tanyashin, V. I., and Murray, N. E., (1977), Molec. Gen., Genet., 156: 203-214). As determined by results of experiments reported in this application, this fragment contained approximately 99% of the gene. Nevertheless, heretofore, it has not been possible to obtain gene 43 in a clonable form.
This is due in part to the fact that straightforward expression of a complete version of gene 43 is inapplicable since T4 DNA polymerase is toxic to bacteria such as E. coli the most likely host. Moreover, using conventional restriction endonuclease procedures, it has not been possible to excise a DNA sequence containing complete gene 43 in a fashion in which it is clonable. Consequently, a need has remained to provide a method enabling the cloning of the DNA sequence encoding T4 DNA polymerase.